If I remember right from Howards articles on MP, the
lipophilic character of the carbocyanines results in poor
cytoplasmic diffusion, hence cytoplasmic MP whilst Rhodamine
goes for mitochondrial MP if the free dye is washed away.
Could that difference in 'clearance' time explain the
differences in delay between depolarization and annexin V
between the groups? Howard also commented on effects of for
example proteins in the medium... changing the staining
kinetic. I don't work on apoptosis yet and don't know the
staining protocols, but I wouldn't be to surprised to find
out that the death of the mitochondria follows the same path
as in bacteria and stands in the beginning of that pathway.
Gerhard Nebe-v.Caron
Unilever Research, Colworth,
Sharnbrook, Bedfordshire
GB - MK44 1LQ
Tel: +44(0)1234-222066
FAX: +44(0)1234-222344
gerhard.nebe-von-caron@unilever.com
______________________________ Reply Separator _________________________________
Subject: Apoptosis detection with Annexin V and DiOC6 -Reply
Author: david_hedley@pmh.toronto.on.ca at INTERNET
Date: 21/01/97 22:43
We have done quite a lot of work on this recently. Mostly we have used
DiIC6(5) to measure mitochondrial membrane potential, since this excites
with a red HeNe laser and allows simultaneous measurements of annexin
V-FITC and indo-1. We use 40nM DiC6(5) for 30 min at 37*, and add
annexin V for the last five min. We add 5?g/ml PI as a live/dead
discriminator at the same time as the annexin V. The DiIC6(5) protocol is
essentially the same as for the more widely used DiOC6(3).
Results for ara-C induced apoptosis in leukemic blasts shows that loss of
mitochondrial membrane potential and phosphatidylserine exposure occur
simultaneously, and precede an increase in ionized calcium. This is
slightly different from the results of Castedo et al (J immunol
1996;157:512-521), where annexin V positivity occurred about an hour
after the mitochondrial membrane changes.
You should check the Castedo paper, since they did dual labelling of
annexin V and mitochondrial membrane potential off an argon laser, using
the new dye CMXRos (available from Molecular Probes). Martin Poot
(who will no doubt also respond to this message) has a recent paper in J
Histochem Cytochem that give more detail about CMXRos.
Hope this helps
David Hedley
Ontario Cancer Institute/Princess Margaret Hospital
Toronto
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